Plants producing 2n gametes or apomeiotic gametes

ABSTRACT

The invention relates to plants wherein the protein OSD1, involved in the transition from meiosis I to meiosis II is inactive. These plants produce Second Division Restitution (SDR) 2n gametes. The invention further relates to plants wherein the inactivation of OSD1 is combined with the inactivation of a gene involved in meiotic recombination in plants, and of a gene involved in the monopolar orientation of the kinetochores during meiosis. These plants produce apomeiotic gametes. These plants are useful in plant breeding.

The invention relates to plants that produce 2n Second DivisionRestitution (SDR) gametes, and to plants that produce apomeioticgametes, and to their use in plant breeding.

2n gametes (also known as diplogametes) are gametes having the somaticchromosome number rather than the gametophytic chromosome number. Theyhave been shown to be useful for the genetic improvement of severalcrops (for review, cf. for instance RAMANNA & JACOBSEN, Euphytica 133,3-18, 2003). In particular, the production of diplogametes allow crossesbetween plants of different ploidy level, for instance crosses betweentetraploid crop plants and their diploid wild relatives, in order to usetheir genetic diversity in plant breeding programs.

The formation of 2n gametes results from anomalies during meiosis (forreview cf. VEILLEUX, Plant Breeding Reviews 3, 252-288, 1985, orBRETAGNOLLE & THOMPSON, New Phytologist 129, 1-22, 1995).

In normal meiosis, chromosomes first duplicate, resulting in pairs ofsister chromatids. This round of replication is followed by two roundsof division, known as meiosis I and meiosis II. During meiosis Ihomologous chromosomes recombine and are separated into two cells, eachof them comprising one entire haploid content of chromosomes. In meiosisII the two cells resulting from meiosis I further divide, and the sisterchromatids segregate. The spores resulting from this division are thushaploid and carry recombined genetic information.

The abnormalities leading to 2n gametes formation include in particularabnormal cytokinesis, the skip of the first or second meiotic division,or abnormal spindle geometry (for review cf. VEILLEUX, Plant BreedingReviews 3, 252-288, 1985, or BRETAGNOLLE & THOMPSON, New Phytologist129, 1-22, 1995). These abnormalities lead to different classes ofunreduced gametes. For instance, failure of the first meiotic divisionresults in First Division Restitution (FDR) gametes, while failure ofthe second meiotic division results in Second Division Restitution (SDR)gametes.

Although numerous mutants able to produce 2n gametes have been reportedin various plant species, only one gene implicated in the formation of2n pollen has been identified and characterized at the molecular leveluntil now. The inactivation of this gene, designated AtPS1 (forArabidopsis thaliana parallel spindles), generates diploid male spores,giving rise to viable diploid pollen grains and to spontaneous triploidplants in the progeny. This gene and its use for producing 2n pollen aredisclosed in European Patent application 08490672, filed on Jul. 8,2008, and in the publication of D′ERFURTH et al (PLoS Genet. 2008November; 4(11):e1000274. Epub 2008 Nov. 28).

The inventors have now identified in the model plant Arabidopsisthaliana, another gene implicated in the formation of 2n gametes inplants. The inventors have found that inactivation of this gene resultsin the skipping of the second meiotic division. This generates diploidmale and female spores, giving rise to viable diploid male and femalegametes, which are SDR gametes. This gene will be hereinafter designatedOSD1, for omission of second division. The sequence of the OSD1 gene ofArabidopsis thaliana is available in the TAIR database under theaccession number At3g57860, or in the GenBank database under theaccession number NM_(—)115648. This gene encodes a protein of 243 aa(GenBank NP_(—)191345), whose sequence is also represented in theenclosed sequence listing as SEQ ID NO: 1.

The OSD1 gene of Arabidopsis thaliana has been previously depicted as“UVI4-Like” gene (UVI4-L), in a publication of HASE et al. (Plant J, 46,317-26, 2006), which describes its paralogue, named UVI4. According toHASE et al. UVI4 acts as a suppressor of endo-reduplication and isnecessary for maintaining the mitotic state whereas OSD1 (UVI4-L) doesnot appear to be required for this process. In contrast, as shownherein, OSD1 appears necessary for allowing the transition from meiosisIto meiosis II.

The inventors have also identified in rice (Oryza sativa) an ortholog ofthe OSD1 gene of Arabidopsis thaliana. The sequence of the OSD1 gene ofOryza sativa is available in the OryGenes or TAIR databases under theaccession number Os02g37850. It encodes a protein of 234 aa, whosesequence is represented in the enclosed sequence listing as SEQ ID NO:35. The OSD1 proteins of Arabidopsis thaliana and Oryza sativa have23.6% identity and 35% similarity over the whole length of theirsequences

The invention thus provides a method for obtaining a plant producingSecond Division Restitution 2n gametes, wherein said method comprisesthe inhibition in said plant of a protein hereinafter designated as OSD1protein, wherein said protein has at least 20%, and by order ofincreasing preference, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 98% sequence identity, or at least 29%, and byorder of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 98% sequence similarity with the AtOSD1 protein ofSEQ ID NO: 1 or with the OsOSD1 protein of SEQ ID NO: 35.

Unless otherwise specified, the protein sequence identity and similarityvalues provided herein are calculated over the whole length of thesequences, using the BLASTP program under default parameters, or theNeedleman-Wunsch global alignment algorithm (EMBOSS pairwise alignmentNeedle tool under default parameters). Similarity calculations areperformed using the scoring matrix BLOSUM62.

The SDR 2n gametes produced according to the invention are useful in allthe usual applications of 2n gametes, for instance for producingpolyploids plants, or to allow crosses between plants of differentploidy level. They can also be useful in methods of genetic mapping, forinstance the method of “Reverse progeny mapping” disclosed in US PatentApplication 20080057583.

The inventors have further found that by combining the inactivation ofOSD1, with the inactivation of two other genes, one (SPO11-1) whichencodes a protein necessary for efficient meiotic recombination inplants, and whose inhibition eliminates recombination and pairing(GRELON et al., Embo J, 20, 589-600, 2001), and another (REC8,At2g47980) which encodes a protein necessary for the monopolarorientation of the kinetochores during meiosis (CHELYSHEVA et al., JCell Sci, 118, 4621-32, 2005), and whose inhibition modifies chromatidsegregation, resulted in a genotype in which meiosis is totally replacedby mitosis without affecting subsequent sexual processes. This genotypewill be called hereinafter MiMe for “mitosis instead of meiosis”. Thisreplacement of meiosis by mitosis results in apomeiotic gametes,retaining all the parent's genetic information (BICKNELL & KOLTUNOW,Plant Cell, 16 Suppl, S228-45, 2004).

FIG. 1 provides a schematic comparison between the mechanisms ofmitosis, normal meiosis, meiosis in the osd1 mutant, meiosis in a mutantlacking SPO11-1 activity (Atspo11-1), meiosis in a double mutant lackingboth SPO11-1 and REC8 activity (Atspo11-1/Atrec8), and meiosis in theMiMe mutant.

During mitosis in diploid cells, chromosomes replicate and sisterchromatids segregate to generate daughter cells that are diploid andgenetically identical to the initial cell. During normal meiosis, tworounds of chromosome segregation follow a single round of replication.At division one, homologous chromosomes recombine and are separated.Meiosis II is more similar to mitosis resulting in equal distribution ofsister chromatids. The obtained spores are thus haploid and carryrecombined genetic information. In the osd1 mutant (this study) meiosisII is skipped giving rise to diploid spores and SDR gametes withrecombined genetic information.

The Atspo11-1 mutant undergoes an unbalanced first division followed bya second division leading to unbalanced spores and sterility.

The Atspo11-1/Atrec8 double mutant undergoes a mitotic-like divisioninstead of a normal first meiotic division, followed by an unbalancedsecond division leading to unbalanced spores and sterility.

In the triple osd1/Atspo11-1/Atrec8 mutant (MiMe), the presence of theAtspo11-1 and Atrec8 mutations leads to a mitotic-like first meioticdivision and the presence of the osd1 mutation prevents the secondmeiotic division from occurring. Thus meiosis is replaced by amitotic-like division. The obtained spores and gametes are geneticallyidentical to the initial cell.

The apomeiotic gametes produced by the MiMe mutant can be used, in thesame way as the SDR 2n gametes, for producing polyploids plants, or forcrossing plants of different ploidy level. They are also of interest forthe production of apomictic plants, i.e plants which are able to formseeds from the maternal tissues of the ovule, resulting in progeny thatare genetic clones of the maternal parent. Although it exists in over400 species of angiosperms, very few crop species are apomictic andattempts to introduce this trait by crossing have failed (SAVIDAN, TheFlowering of Apomixis: From Mechanisms to Genetic Engineering 2001;SPILLANE et al., Sexual Plant Reproduction, 14, 2001).

A further object of the present invention is thus a method for obtaininga plant producing apomeiotic gametes, wherein said method comprises theinhibition in said plant of the following proteins:

-   -   a) an OSD1 protein as defined above;    -   b) a protein involved in initiation of meiotic recombination in        plants, said protein being selected among:        -   i) a protein hereinafter designated as SPO11-1 protein,            wherein said protein has at least 40%, and by order of            increasing preference, at least 45, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95 or 98% sequence identity, or at least 60%,            and by order of increasing preference, at least, 65, 70, 75,            80, 85, 90, 95 or 98% sequence similarity with the SPO11-1            protein of SEQ ID NO: 2;        -   ii) a protein hereinafter designated as SPO11-2 protein,            wherein said protein has at least 40%, and by order of            increasing preference, at least 45, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95 or 98% sequence identity, or at least 60%,            and by order of increasing preference, at least, 65, 70, 75,            80, 85, 90, 95 or 98% sequence similarity with the SPO11-2            protein of SEQ ID NO: 3;        -   iii) a protein hereinafter designated as PRD1 protein,            wherein said protein has at least 25%, and by order of            increasing preference, at least 30, 35, 40, 45, 50, 55, 60,            65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at            least 35%, and by order of increasing preference, at least,            40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98%            sequence similarity with the PRD1 protein of SEQ ID NO: 4;        -   iv) a protein hereinafter designated as PAIR1 protein,            wherein said protein has at least 30%, and by order of            increasing preference, at least 35, 40, 45, 50, 55, 60, 65,            70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least            40%, and by order of increasing preference, at least, 45,            50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence            similarity with the PAIR1 protein of SEQ ID NO: 5;    -   c) a protein hereinafter designated as Rec8 protein, wherein        said protein has at least 40%, and by order of increasing        preference, at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95        or 98% sequence identity, or at least 45%, and by order of        increasing preference, at least, 50, 55, 60, 65, 70, 75, 80, 85,        90, 95 or 98% sequence similarity with the Rec8 protein of SEQ        ID NO: 6.

SEQ ID NO: 2 represents the sequence of the SPO11-1 protein ofArabidopsis thaliana. This sequence is also available in the Swissprotdatabase under the accession number Q9M4A2.

SEQ ID NO: 3 represents the sequence of the SPO11-2 protein ofArabidopsis thaliana. This sequence is also available in the SwissProtdatabase under the accession number Q9M4A1.

SEQ ID NO: 4 represents the sequence of the PRD1 protein of Arabidopsisthaliana. This sequence is also available in the GenBank database underthe accession number ABQ12642.

SEQ ID NO: 5 represents the sequence of the PAIR1 protein of Arabidopsisthaliana. This sequence is also available in the GenBank database underthe accession number NP_(—)171675.

SEQ ID NO: 6 represents the sequence of the Rec8 protein of Arabidopsisthaliana. This sequence is also available in the GenBank database underthe accession number NP_(—)196168.

The SPO11-1, SPO11-2, PRD1, PAIR1, and Rec8 proteins are conserved inhigher plants, monocotyledons as well as dicotyledons. By way ofnon-limitative examples of orthologs of SPO11-1, SPO11-2, PRD1, PAIR1and Rec8 proteins of Arabidopsis thaliana in monocotyledonous plants,one can cite the Oryza sativa SPO11-1, SPO11-2, PRD1, PAIR1, and Rec8proteins. The sequence of the Oryza sativa SPO11-1 protein is availablein GenBank under the accession number AAP68363; the sequence of theOryza sativa SPO11-2 protein is available in GenBank under the accessionnumber NP_(—)001061027; the sequence of the Oryza sativa PRD1 protein isavailable in GenBank under the accession number EAZ30311; the sequenceof the Oryza sativa PAIR1 protein is available in SwissProt under theaccession number Q75RY2; the sequence of the Oryza sativa Rec8 proteinis available in GenBank under the accession number AAQ75095.

The inhibition of the above mentioned OSD1, SPO11-1, SPO11-2, PRD1,PAIR1, or Rec8 proteins can be obtained either by abolishing, blocking,or decreasing their function, or advantageously, by preventing ordown-regulating the expression of the corresponding genes.

By way of example, inhibition of said protein can be obtained bymutagenesis of the corresponding gene or of its promoter, and selectionof the mutants having partially or totally lost the activity of saidprotein. For instance, a mutation within the coding sequence can induce,depending on the nature of the mutation, the expression of an inactiveprotein, or of a protein with impaired activity; in the same way, amutation within the promoter sequence can induce a lack of expression ofsaid protein, or decrease thereof.

Mutagenesis can be performed for instance by targeted deletion of thecoding sequence or of the promoter of the gene encoding said protein orof a portion thereof, or by targeted insertion of an exogenous sequencewithin said coding sequence or said promoter. It can also be performedby inducing random mutations, for instance through EMS mutagenesis orrandom insertional mutagenesis, followed by screening of the mutantswithin the desired gene. Methods for high throughput mutagenesis andscreening are available in the art. By way of example, one can mentionTILLING (Targeting Induced Local Lesions IN Genomes, described byMcCallum et al., 2000).

Among the mutations within the OSD1 gene, those resulting in the abilityto produce SDR 2n gametes can be identified on the basis of thephenotypic characteristics of the plants which are homozygous for thismutation: these plants can form at least 5%, preferably at least 10%,more preferably at least 20%, still more preferably at least 50%, and upto 100% of dyads as a product of meiosis.

Among the mutations within a gene encoding a protein involved ininitiation of meiotic recombination in plants, such as the SPO11-1 geneor the SPO11-2, PRD1, or PAIR1 gene, those useful for obtaining a plantproducing apomeiotic gametes can be identified on the basis of thephenotypic characteristics of the plants which are homozygous for thismutation, in particular the presence of univalents instead of bivalentsat meiosis I, and the sterility of the plant.

Among the mutants having a mutation within the REC8 gene, those usefulfor obtaining a plant producing apomeiotic gametes can be identified onthe basis of the phenotypic characteristics of the plants which arehomozygous for this mutation, in particular chromosome fragmentation atmeiosis, and sterility of the plant.

According to a preferred embodiment of the method of the invention forobtaining a plant able to produce SDR 2n gametes, said method comprises:

a) providing a plant having a mutation within an allele of the OSD1 generesulting in the inhibition of the protein encoded by this allele, saidplant being heterozygous for this mutation;

b) self fertilizing said plant of step a) in order to obtain a planthomozygous for said mutation.

According to a preferred embodiment of the method of the invention forobtaining a plant able to produce apomeiotic gametes, said methodcomprises:

a) providing a plant having a mutation within an allele of the OSD1 generesulting in the inhibition of the protein encoded by this allele, saidplant being heterozygous for this mutation;

b) providing a plant having a mutation within an allele of a geneselected among the SPO11-1, SPO11-2, PRD1, or PAIR1 gene resulting inthe inhibition of the protein encoded by said allele, said plant beingheterozygous for this mutation;

c) providing a plant having a mutation within an allele of the REC8 generesulting in the inhibition of the protein encoded by said allele, saidplant being heterozygous for this mutation;

e) crossing the plants of steps a) b) and c) in order to obtain a planthaving a mutation within an allele of the OSD1 gene, a mutation withinan allele of a gene selected among the SPO11-1, SPO11-2, PRD1, or PAIR1gene, and a mutation within an allele of the REC8 gene, said plant beingheterozygous for each mutation;

f) self fertilizing the plant of step e) in order to obtain a planthomozygous for the mutation within the OSD1 gene, for the mutationwithin the gene selected among the SPO11-1, SPO11-2, PRD1, or PAIR1gene, and for the mutation within the REC8 gene.

Alternatively, the inhibition of the target protein is obtained bysilencing of the corresponding gene. Methods for gene silencing inplants are known in themselves in the art. For instance, one can mentionby antisense inhibition or co-suppression, as described by way ofexample in U.S. Pat. Nos. 5,190,065 and 5,283,323. It is also possibleto use ribozymes targeting the mRNA of said protein.

Preferred methods are those wherein gene silencing is induced by meansof RNA interference (RNAi), using a silencing RNA targeting the gene tobe silenced. Various methods and DNA constructs for delivery ofsilencing RNAs are available in the art.

A “silencing RNA” is herein defined as a small RNA that can silence atarget gene in a sequence-specific manner by base pairing tocomplementary mRNA molecules. Silencing RNAs include in particular smallinterfering RNAs (siRNAs) and microRNAs (miRNAs).

Initially, DNA constructs for delivering a silencing RNA in a plantincluded a fragment of 300 bp or more (generally 300-800 bp, althoughshorter sequences may sometime induce efficient silencing) of the cDNAof the target gene, under transcriptional control of a promoter activein said plant. Currently, the more widely used silencing RNA constructsare those that can produce hairpin RNA (hpRNA) transcripts. In theseconstructs, the fragment of the target gene is inversely repeated, withgenerally a spacer region between the repeats (for review, cf. WATSON etal., 2005). One can also use artificial microRNAs (amiRNAs) directedagainst the gene to be silenced (for review about the design andapplications of silencing RNAs, including in particular amiRNAs, inplants cf. for instance OSSOWSKI et al., (Plant J., 53, 674-90, 2008).

The present invention provides tools for silencing one or more targetgene(s) selected among OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, and REC8,including in particular expression cassettes for hpRNA or amiRNAtargeting said gene (s).

An expression cassette of the invention may comprise for instance:

-   -   a promoter functional in a plant cell;    -   one or more DNA construct(s) of 200 to 1000 bp, preferably of        300 to 900 bp, each comprising a fragment of a cDNA of a target        gene selected among OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, and        REC8, or of its complementary, or having at least 95% identity,        and by order of increasing preference, at least 96%, 97%, 98%,        or 99% identity with said fragment, said DNA construct(s) being        placed under transcriptional control of said promoter.

According to a preferred embodiment of the invention, an expressioncassette for hpRNA comprises:

-   -   a promoter functional in a plant cell,    -   one or more hairpin DNA construct(s) capable, when transcribed,        of forming a hairpin RNA targeting a gene selected among OSD1,        SPO11-1, SPO11-2, PRD1, PAIR1, and REC8;

said DNA construct(s) being placed under transcriptional control of saidpromoter.

Generally, said hairpin DNA construct comprises: i) a first DNA sequenceof 200 to 1000 bp, preferably of 300 to 900 bp, consisting of a fragmentof a cDNA of the target gene, or having at least 95% identity, and byorder of increasing preference, at least 96%, 97%, 98%, or 99% identitywith said fragment; ii) a second DNA sequence that is the complementaryof said first DNA, said first and second sequences being in oppositeorientations and ii) a spacer sequence separating said first and secondsequence, such that these first and second DNA sequences are capable,when transcribed, of forming a single double-stranded RNA molecule. Thespacer can be a random fragment of DNA. However, preferably, one willuse an intron which is spliceable by the target plant cell. Its size isgenerally 400 to 2000 nucleotides in length.

According to another preferred embodiment of the invention, anexpression cassette for an amiRNA comprises:

-   -   a promoter functional in a plant cell,    -   one or more DNA construct(s) capable, when transcribed, of        forming an amiRNA targeting a gene selected among OSD1, SPO11-1,        SPO11-2, PRD1, PAIR1, and REC8;

said DNA construct(s) being placed under transcriptional control of saidpromoter.

Advantageously, an expression cassette of the invention comprises a DNAconstruct targeting the OSD1 gene. According to a particularly preferredembodiment it comprises: a DNA construct targeting the OSD1 gene, a DNAconstruct targeting a gene selected among SPO11-1, SPO11-2, PRD1, andPAIR1, and a DNA construct targeting REC8.

A large choice of promoters suitable for expression of heterologousgenes in plants is available in the art.

They can be obtained for instance from plants, plant viruses, orbacteria such as Agrobacterium. They include constitutive promoters,i.e. promoters which are active in most tissues and cells and under mostenvironmental conditions, as well as tissue-specific or cell-specificpromoters which are active only or mainly in certain tissues or certaincell types, and inducible promoters that are activated by physical orchemical stimuli, such as those resulting from nematode infection.

Non-limitative examples of constitutive promoters that are commonly usedin plant cells are the cauliflower mosaic virus (CaMV) 35S promoter, theNos promoter, the rubisco promoter, the Cassaya vein Mosaic Virus(CsVMV) promoter.

Organ or tissue specific promoters that can be used in the presentinvention include in particular promoters able to confermeiosis-associated expression, such as the DMC1 promoter (KLIMYUK &JONES, Plant J, 11, 1-14, 1997); one can also use any of the endogenouspromoters of the genes OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, or REC8.

The DNA constructs of the invention generally also include atranscriptional terminator (for instance the 35S transcriptionalterminator, or the nopaline synthase (Nos) transcriptional terminator).

The invention also includes recombinant vectors containing a chimericDNA construct of the invention. Classically, said recombinant vectorsalso include one or more marker genes, which allow for selection oftransformed hosts.

The selection of suitable vectors and the methods for inserting DNAconstructs therein are well known to persons of ordinary skill in theart. The choice of the vector depends on the intended host and on theintended method of transformation of said host. A variety of methods forgenetic transformation of plant cells or plants are available in the artfor many plant species, dicotyledons or monocotyledons. By way ofnon-limitative examples, one can mention virus mediated transformation,transformation by microinjection, by electroporation, microprojectilemediated transformation, Agrobacterium mediated transformation, and thelike.

The invention also provides a host cell comprising a recombinant DNAconstruct of the invention. Said host cell can be a prokaryotic cell,for instance an Agrobacterium cell, or a eukaryotic cell, for instance aplant cell genetically transformed by a DNA construct of the invention.The construct may be transiently expressed; it can also be incorporatedin a stable extrachromosomal replicon, or integrated in the chromosome.

According to a preferred embodiment of the method of the invention forproviding a plant able to produce SDR 2n gametes, said plant is atransgenic plant, and said method comprises:

a) transforming at least one plant cell with a vector containing a DNAconstruct of the invention targeting the OSD1 gene;

b) cultivating said transformed plant cell in order to regenerate aplant having in its genome a transgene containing said DNA construct.

According to a preferred embodiment of the method of the invention forobtaining a plant able to produce apomeiotic gametes, said plant is atransgenic plant, and said method comprises:

a) transforming at least one plant cell with a vector containing a DNAconstruct of the invention targeting the OSD1 gene, a vector containinga DNA construct of the invention targeting a gene selected amongSPO11-1, SPO11-2, PRD1, and PAIR1, and a vector containing a DNAconstruct of the invention targeting the REC8 gene;

b) cultivating said transformed plant cell in order to regenerate aplant having in its genome transgenes containing said DNA constructs.

According to another preferred embodiment of the method of the inventionfor obtaining a plant able to produce apomeiotic gametes, said plant isa transgenic plant, and said method comprises:

a) transforming at least one plant cell with a vector containing a DNAconstruct of the invention targeting the OSD1 gene, a DNA construct ofthe invention targeting a gene selected among SPO11-1, SPO11-2, PRD1,and PAIR1, and a vector containing a DNA construct of the inventiontargeting the REC8 gene;

b) cultivating said transformed plant cell in order to regenerate aplant having in its genome a transgene containing said DNA constructs.

The invention also encompasses plants able to produce SDR 2n gametes orapomeiotic gametes, obtainable by the methods of the invention.

This includes in particular plants comprising:

-   -   a mutation within the OSD1 gene, wherein the OSD1 protein is        inhibited as a result of this mutation; and    -   a mutation within a gene selected among SPO11-1, SPO11-2, PRD1,        or PAIR1 gene, wherein the SPO11-1, SPO11-2, PRD1, or PAIR1        protein encoded by said gene is inhibited as a result of this        mutation; and    -   a mutation within the REC8 gene, wherein the Rec8 protein is        inhibited as a result of this mutation.

This also includes plants genetically transformed by one or more DNAconstruct(s) of the invention. Preferably, said plants are transgenicplants, wherein said construct is contained in a transgene integrated inthe plant genome, so that it is passed onto successive plantgenerations.

The expression of a chimeric DNA construct targeting the OSD1 gene,resulting in a down regulation of the OSD1 protein, provides to saidtransgenic plant the ability to produce 2n SDR gametes. Theco-expression of a chimeric DNA construct targeting the OSD1 gene, achimeric DNA construct targeting a gene selected among SPO11-1, SPO11-2,PRD1, and PAIR1, and a chimeric DNA construct targeting the REC8 gene,results in a down regulation of the proteins encoded by these threegenes and provides to said transgenic plant the ability to produceapomeiotic gametes.

The invention also encompasses a method for producing SDR 2n gametes,wherein said method comprises cultivating a plant obtainable by a methodof the invention and recovering the gametes produced by said plant.Preferably said gametes comprises at least 10%, more preferably at least20%, and by order of increasing preference, at least 30%, 40%, 50%, 60%,70%, 80%, or 90% of viable 2n gametes.

The invention also encompasses a method for producing apomeioticgametes, wherein said method comprises cultivating a plant obtainable bya method of the invention and recovering the gametes produced by saidplant. Preferably said gametes comprises at least 10%, more preferablyat least 20%, and by order of increasing preference, at least 30%, 40%,50%, or 60%, 70%, 80%, or 90% of viable apomeiotic gametes.

The present invention applies to a broad range of monocot- ordicotyledon plants of agronomical interest. By way of non-limitativeexamples, one can mention potato, rice, wheat, maize, tomato, cucumbers,alfafa, sugar cane, sweet potato, manioc, clover, soybean, ray-grass,banana, melon, watermelon, cotton or ornamental plants such as roses,lilies, tulips, and narcissus.

Foregoing and other objects and advantages of the invention will becomemore apparent from the following detailed description and accompanyingdrawings. It is to be understood however that this foregoing detaileddescription is exemplary only and is not restrictive of the invention.

EXAMPLES Experimental Procedures Plant Material and Growth Conditions.

Arabidopsis plants were cultivated as described in VIGNARD et al., (PLoSGenet, 3, 1894-906, 2007). For germination assays and cytometryexperiments Arabidopsis were cultivated in vitro on Arabidopsis medium(ESTELLE & SOMERVILLE, Mol. Gen. Genet., 206, 200-06, 1987) at 21° C.with a 16 h day/8 h night photoperiod and 70% hygrometry.

Genetic Analysis.

Plants were genotyped by PCR (30 cycles of 30s at 94° C., 30s at 56° C.and 1 min at 72° C.) using two primer pairs. For each genotype theprimer pair is shown in Table I and the primer pair specific to theinsertion is shown in Table II.

TABLE I Primers for Wild-type allele osd1-1pst15307U (5′CGTCACTCTCCCCAAGAAAG 3′) (SEQ ID NO: 7)pst15307L (5′GGCTAAGCAAGCCTGCTATG 3′) (SEQ ID NO: 8) osd1-2GT21481U (5′CCGGTGTTCTTGTGACTCG 3′) (SEQ ID NO: 9)GT21481L (5′GCAGATTCCTAATTCAGCTC 3′) (SEQ ID NO: 10) Atspo11-1-3N646172U (5′ AATCGGTGAGTCAGGTTTCAG 3′) (SEQ ID NO: 11) N646172L (5′CCATGGATGAAAGCGATTTAG 3′) (SEQ ID NO: 12) Atrec8-3N836037U (5′CTCATATTCACGGTGCTCCC 3′) (SEQ ID NO: 13)N836037L (5′GGGGGAAAAGAGAAAGGTTC 3′) (SEQ ID NO: 14)

TABLE II Primers for mutant allele osd1-1 pst15307LDs5-2a (5′TCCGTTCCGTTTTCGTTTTTTAC3′) (SEQ ID NO: 15) osd1-2 GT21481UDs3-4 (5′CCGTCCCGCAAGTTAAATATG3′) (SEQ ID NO: 16) Atspo11-1-3 N646172LLbSalk2 (5′ GCTTTCTTCCCTTCCTTTCTC 3′) (SEQ ID NO: 17) Atrec8-3 N836137LLB3sail (5′TAGCATCTGAATTTCATAACCAATCTCGATACAC3′) (SEQ ID NO: 18)

Genetic markers used to genotype the osd1-1(No-0)/osd1-2(Ler)×Col-0 F1population and osd1-1(No-0)/spo11-1(Col-0)1rec8(Col-0) triple mutant×LerF1 population are listed in Table III. The PCR conditions were 40 cyclesof 30s at 94° C., 30s at Tm and 30s at 72° C.

TABLE III marker Chrom. Position pb Primer 1 (SEQ ID NO:)Primer 2 (SEQ ID NO:) Msat1-13 1 25827433 CAACCACCAGGCTC (19)GTCAAACCAGTTCAATCA(20) F5i14 1 24374008 CTGCCTGAAATTGTCGAAAC(21)GGCATCACAGTTCTGATTCC(22) Msat2-18 2  2799644 TAGTCTCTTTTGGTGCGCATA(23)AGCCTCTCCAAGCTTAGGTCT(24) Msat2-21 2 11461020 ATTTTTAGCCCAATCACGTTT(25)AGGTCAAGTGAAAGGGTAAGG(26) Msat2-9 2 18152580 TAAAAGAGTCCCTCGTAAAG(27)GTTGTTGTTGTGGCATT(28) CapsK4_10355 4 10354800 ACCCATTTGGTGATGCTAAC(29)GAGCAGTTTCCACTTTGTCC(30) Msat4-18 4 11966304 TGTAAATATCGGCTTCTAAG(31)CTGAAACAAATCGCATTA(32) Nga151 5  4669932 GTTTTGGGAAGTTTTGCTGG(33)CAGTCTAAAAGCGAGAGTATGATG(34)

These markers were amplified (40 cycles of 30s at 94° C., 30s at 58° C.and 30s at 72° C.) with the indicated primers and observed aftermigration on 3% agarose gel.

CAPS K4 10355 was observed after Eco47III/HpaII double digestion. Thetwo primer pairs specific for the osd1-1 and osd1-2 insertion borderswere used as a marker on chromosome 3.

Cytology and Flow Cytometry:

Final meiotic products were observed as described in AZUMI et al., (EmboJ, 21, 3081-95., 2002) and viewed with a conventional light microscopewith a 40× dry objective. Chromosomes spreads and observations werecarried out using the technique described in MERCIER et al., (Biochimie,83, 1023-28, 2001). The DNA fluorescence of spermatic pollen nuclei wasquantified using open LAB 4.0.4 software. For each nucleus thesurrounding background was calculated and subtracted from the globalfluorescence of the nucleus. Meiotic spindles were observed according tothe protocol described in MERCIER et al., (Genes Dev, 15, 1859-71, 2001)except that the DNA was counter-stained with DAPI. Observations weremade using an SP2 Leica confocal microscope. Images were acquired with a63× water objective in xyz and 3D reconstructions were made using Leicasoftware. Projections are shown. Cells were imaged at excitation 488 nmand 405 nm with AlexaFluor488 and DAPI respectively. Arabidopsis genomesizes were measured as described in MARIE & BROWN, (Biol Cell, 78,41-51, 1993) using tomato Lycopersicon esculentum cv “Montfavet” as thestandard. (2C=1.99 pg, % GC-40.0%)

Example 1 Production of Diploid Gametes by osd1 Mutants

As a part of an expression profiling screen for meiotic genes, using theExpression Angler tool (TOUFIGHI et al., Plant J, 43, 153-63, 2005) withthe AtGenExpress tissue set (SCHMID et al., Nat Genet, 37, 501-6, 2005),At3g57860 was selected as a good candidate due to its co-regulation withseveral known meiotic genes. At3g57860 corresponds to the UVI4-Like gene(UVI4-L) which was briefly described in a study of its paralogue, theUVI4 gene (HASE et al., Plant J, 46, 317-26, 2006). Due to its role inmeiosis (see below) we renamed the At3g57860 gene OSD1, for omission ofsecond division. The OSD1 and UVI4 proteins are conserved throughout theplant kingdom but do not contain any obvious conserved known functionaldomains. No homologues were identified outside the plant kingdom.

We investigated the role of the OSD1 gene by isolating andcharacterising two mutants. The osd1-1 (pst15307) and the osd1-2(GT21481) Ds insertional mutants are in the Nooseen (No-0) and Landsberg(Ler) backgrounds, respectively, and in both cases the insertion is inthe second exon of the OSD1 gene.

The intron/exon structure of the OSD1 gene and the location of the twodifferent Ds insertions are shown in FIG. 2. The OSD1 gene contains 3exons and 2 introns and encodes a protein of 243 amino acids. Thepositions of the two Ds insertions are indicated by triangles.

FIG. 3 represents meiosis in wild-type plants and FIG. 4 representsmeiosis in osd1 mutants.

Legend of FIG. 3: (A) Pachytene. Homologous chromosomes are fullysynapsed. (B) Diakinesis. Five pairs of homologous chromosomes(bivalent), linked by chiasmata, are observed. (C) Metaphase I. The fivebivalent are aligned on the metaphase plate. (D) Anaphase I. Thehomologous chromosomes are separated. (E) Telophase I. (F) Metaphase II.The pairs of sister chromatids align on the metaphase plates. (G)Anaphase II. The sister chromatids are separated. (H and I) TelophaseII. Four haploid spores are formed (tetrad). Scale bar=10 μm

Legend of FIG. 4: (A and B) Male meiotic products stained with toluidineblue. (A) A wild type tetrad. (B) A dyad in the osd1-1 mutant. (C to D)Male meiosis in osd1 is indistinguishable from wild type until telophaseI (compare to FIG. 3), but no figures characteristic of a seconddivision were observed. (C) pachytene. (D) diakinesis. (E) metaphase I.(F) Anaphase I. (G) Telophase I. (H) Metaphase I of female meiosis inosd1.

In both independent osd1 mutants the products of male meiosis were dyads(osd1-1: 714/714 osd1-2: 334/334) instead of tetrads (FIGS. 4A and B).Complementation tests between osd1-1 and osd1-2 confirmed that thesemutations are allelic (osd1-1/osd1-2: 369 dyads/369), and thusdemonstrated that the observed dyads are due to disruption of the OSD1gene. Osd1 mutants did not show any somatic developmental defects, maleand female gametophyte lethality or reduced fertility (wild type 38±11seeds/fruit, osd1 35±6).

Next, we measured ploidy levels among the offspring of diploid osd1mutants. Among selfed progeny, tetraploids (84%) and triploids (16%),but no diploid plants were found (osd1-1: n=56; osd1-2: n=24). Whenmutant pollen was used to fertilise a wild type plant, all the resultingprogeny were triploid (osd1-1: n=75). When mutant ovules were fertilisedwith wild type pollen grains we isolated 12% diploid and 88% triploidplants (n=25). This demonstrated that the osd1 mutants produce highlevels of male (100%) and female (˜85%) diploid spores, which result infunctional gametes.

To unravel the mechanisms leading to dyad production in osd1, weinvestigated chromosome behaviour during meiosis. Both male and femalemeiosis I were indistinguishable from wild type (compare FIG. 4 withFIG. 3). Notably, chiasmata, the cytological manifestation ofcrossovers, and bivalents were observed. However, we were unable to findany meiosis II figures (among >500 male meiocytes from prophase to sporeformation), strongly suggesting that dyad production is due to anabsence of the second meiotic division. If this second division does nottake place then any heterozygosis at centromeres will be lost in thediploid gametes because of sister chromatids co-segregation andhomologues separation during the first division. Because ofrecombination, any loci which are not linked to centromeres willsegregate. We tested our assumption by taking advantage of the twodifferent genetic backgrounds of the osd1-1 (No-0) and osd1-2 mutants(Ler). F1 plants bearing the two mutations—mutant for osd1 andheterozygous for any No-0/Ler polymorphisms—were crossed as male orfemale to a third genetic background, Columbia (Col-0). Karyotyping andgenotyping of the obtained plants for trimorphic molecular markersprovided direct information on the genetic make-up of pollen grains andfemale gametophytes produced by the mutant. All the diploid gametestested had the predicted genetic characteristics. They weresystematically homozygous at centromeres and segregating—because ofrecombination—at other loci (n=48 for male diploid gametes and n=41 forfemale diploid gametes). These results confirmed that the absence of asecond meiotic division is indeed the cause of 2n gametes production inosd1. This mechanism also implies that unbalanced chromosome segregationat meiosis I would give rise to unbalanced dyads in osd1; this wasconfirmed by analysing a double Atspo11-1/osd1-1 mutant (data notshown).

Due to an absence of the second meiotic division, osd1 mutants producehigh frequencies of viable diploid male and female gametophytes, whichgenerate, after fecundation, viable tetraploid plants. However, thisphenomenon differs from apomeiosis in that the produced gametes aregenetically different from the mother plant.

Example 2 Production of Apomeiotic Gametes by Tripleosd1/Atrec8/Atspo11-1 Mutants

In double Atspo11-1/Atrec8 mutants the first meiotic division isreplaced by a mitotic-like division, followed by an unbalanced seconddivision which leads to unbalanced spores and sterility (CHELYSHEVA etal., J Cell Sci, 118, 4621-32, 2005).

We generated osd1/Atrec8/Atspo11-1 mutants. Plants heterozygous for bothAtspo11-1 and Atrec8 mutations were obtained by crossing plantsheterozygous for each mutation, and were crossed by a plant heterozygousfor osd1. Triple heterozygous plants identified were self-fertilized andplants homozygous for the three mutations were analyzed.

Observation of chromosome behaviour during male and female meiosis ofthese mutants is shown in FIG. 5.

Legend of FIG. 5: (A) Male metaphase I (B) Male anaphase I. The vignetteshows a dyad in MiMe. (C) Female metaphase I. (D) Female anaphase I.Scale bar=10 μm.

These observations revealed a mitotic-like division: 10 univalentsaligned on the metaphase plate and sister chromatids separated atanaphase (FIG. 5).

The Atspo11-1 and Atrec8 mutations lead to a mitotic-like first meioticdivision and the osd1 mutation prevents the second meiotic division fromtaking place. This results in replacement of meiosis by a mitotic-likedivision, and in apomeiosis.

We called this genotype MiMe for “mitosis instead of meiosis”. MiMeplants generate dyads (408/408) and are fertile (25±6 seeds per fruit).The osd1 mutation therefore suppressed the sterility phenotype of theAtspo11-1/Atrec8 double mutant.

The selfed progeny of MiMe plants were systematically tetraploid (n=24)and backcrosses between diploid MiMe plants and wild type plantsgenerated triploid plants regardless of whether male (n=24) or female(n=67) MiMe gametes were used, showing that this mitotic-like divisiongives rise to functional diploid gametes. All the gametes (male andfemale), tested similarly as described above, systematically retainedthe mother plant heterozygosity for every genetic marker tested and werethus genetically identical to the mother plant. These results confirmthat MiMe plants undergo a mitotic-like division instead of a normalmeiotic division, without affecting subsequent sexual processes.

When meiosis is replaced by mitosis ploidy is expected to double witheach generation. This was observed in MiMe plants, as shown in FIG. 6.

Legend of FIG. 6: Left column: mitotic metaphase, scale bar=10 μm. Rightcolumns: the corresponding four weeks old plants, (scale bar=2 cm) andflowers (scale bar=1 mm)

In subsequent generations, we obtained tetraploid (4N, 20 chromosomes,n=26) and octoploid (8N, 40 chromosomes, n=33).

Example 3 Identification of a Rice Ortholog of the Arabidopsis OSD1 Gene

The Oriza sativa genome contains two OSD1/UVI4 homologue candidates(Os02g37850 and Os04g39670). We isolated two T-DNA insertion mutants inone of this putative homologue (Os02g37850). The two lines, AMBA12 andAMQF10 were genotyped by PCR to select homozygotes. In both lines weobserved spontaneous tetraploids plants among the offspring of diploidmutant plants, suggestive of the production of functional male andfemale 2n gametes (AMBA 12: 100% of tetraploid, n=30; AMQF10 37% oftetraploids, n=27). We then studied the meiotic products in AMB12mutants (n>400) and observed the production of 100% of dyads instead oftetrads, as illustrated by FIG. 7.

Legend of FIG. 7: A: Tetrad of spores in wild type; B: Dyad of spores inAMB12.

This phenotype is identical to the Arabidopsis osd1 mutant. To unravelthe mechanisms leading to dyad production in AMBA12 homozygote mutants,we investigated chromosome behavior during meiosis. Meiosis I wasindistinguishable from wild type. Notably, chiasmata, the cytologicalmanifestation of crossovers, and bivalents were observed. However, wewere unable to find any meiosis II figures, strongly suggesting that 2Nspores production is due to an absence of the second meiotic division,like in Arabidopsis osd1. Altogether, these results show that Os02g37850is the functional homologue of Arabidopsis OSD1 and therefore called itOsOSD1. OSD1 and OsOSD1 proteins have 23.6% identity and 35% similarityon an alignment that covers the whole length of the sequences (EMBOSSpairwise alignment Needle tool).

1. A method for obtaining a plant producing Second Division Restitution2n gametes, wherein said method comprises the inhibition in said plantof a protein hereinafter designated as OSD1 protein, wherein saidprotein has at least 20° A) sequence identity, or at least 29% sequencesimilarity with the AtOSD1 protein of SEQ ID NO: 1, or with the OsOSD1protein of SEQ ID NO:
 35. 2. A method according to claim 1, whereininhibition of the OSD1 protein is obtained by mutagenesis of the OSD1gene or of its promoter, and the mutants having partially or totallylost the OSD1 protein activity are selected.
 3. A method according toclaim 1, wherein the inhibition of the OSD1 protein is obtained byexpressing in said plant a silencing RNA targeting the gene encodingsaid protein.
 4. A method for obtaining a plant producing apomeioticgametes, wherein said method comprises the inhibition in said plant ofthe following proteins: a) an OSD1 protein as defined in claim 1; b) aprotein involved in initiation of meiotic recombination in plants, saidprotein being selected among: i) a protein hereinafter designated asSPO11-1 protein, wherein said protein has at least 40% sequenceidentity, or at least 60% sequence similarity with the SPO11-1 proteinof SEQ ID NO: 2; ii) a protein hereinafter designated as SPO11-2protein, wherein said protein has at least 40% sequence identity, or atleast 60% sequence similarity with the SPO11-2 protein of SEQ ID NO: 3;iii) a protein hereinafter designated as PRD1 protein, wherein saidprotein has at least 25% sequence identity, or at least 35% sequencesimilarity with the prd1 protein of SEQ ID NO: 4; or iv) a proteinhereinafter designated as PAIR1 protein, wherein said protein has atleast 30% sequence identity, or at least 40% sequence similarity withthe PAIR1 protein of SEQ ID NO: 5; c) a protein hereinafter designatedas Rec8 protein, wherein said protein has at least 40% sequenceidentity, or at least 45% sequence similarity with the Rec8 protein ofSEQ ID NO:
 6. 5. A method according to claim 4, wherein inhibition of atleast one of the OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, or Rec8 proteinsis obtained by mutagenesis of the gene encoding said protein of or ofits promoter, and the mutants having partially or totally lost theactivity of said protein are selected.
 6. A method according to claim 4,wherein inhibition of at least one of the OSD1, SPO11-1, SPO11-2, PRD1,PAIR1, or Rec8 proteins is obtained by expressing in said plant asilencing RNA targeting the gene encoding said protein.
 7. An expressioncassette comprising: a promoter functional in a plant cell; at least oneDNA construct selected among: a) one or more DNA construct(s) of 200 to1000 bp, each comprising a fragment of a cDNA of a target gene selectedamong OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, and REC8, or of itscomplementary, or having at least 95% identity with said fragment, saidDNA sequence(s) being placed under transcriptional control of saidpromoter. b) one or more hairpin DNA construct(s) capable, whentranscribed, of forming a hairpin RNA targeting a gene selected amongOSD1, SPO11-1, SPO11-2, PRD1, PAIR1, and REC8; or c) one or more DNAconstruct(s) capable, when transcribed, of forming an amiRNA targeting agene selected among OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, and REC8; saidDNA construct(s) being placed under transcriptional control of saidpromoter.
 8. An expression cassette of claim 7 comprising a DNAconstruct targeting the OSD1 gene.
 9. An expression cassette of claim 7,comprising: a DNA construct targeting the OSD1 gene, a DNA constructtargeting a gene selected among SPO11-1, SPO11-2, PRD1, and PAIR1, or aDNA construct targeting REC8.
 10. A recombinant vector comprising anexpression cassette of claim
 7. 11. A plant producing Second DivisionRestitution 2n gametes, which is a transgenic plant containing atransgene comprising an expression cassette of claim
 8. 12. A plantproducing apomeiotic gametes, obtainable by the method of claim
 4. 13. Aplant producing apomeiotic gametes, which is a transgenic plantcontaining a transgene comprising an expression cassette of claim
 9. 14.A method for producing Second Division Restitution 2n gametes, whereinsaid method comprises cultivating a plant obtainable by a method ofclaim 1, and recovering the gametes produced by said plant.
 15. A methodfor producing apomeiotic gametes, wherein said method comprisescultivating a plant obtainable by a method of claim 4, and recoveringthe gametes produced by said plant.
 16. A recombinant vector comprisingan expression cassette of claim
 8. 17. A recombinant vector comprisingan expression cassette of claim 9.